Geographic variation in acid stress tolerance of the moor frog,Rana arvalis. II. Adaptive maternal effects

Abstract The knowledge about the relative contributions of additive genetic and maternal effects, as well as the proximate determinants of maternal effects variation, on population differentiation remains elusive. Likewise, although embryonic performance is often an important component of fitness, it has been relatively little explored in respect to population differentiation. By conducting reciprocal crosses between an acid and a neutral origin population of moor frogs ( Rana arvalis ), we investigated the relative importance of additive genetic versus maternal effects in local adaptation to acidity in embryonic traits. Furthermore, by performing removal experiments of gelatinous egg capsules (jelly), we evaluated the possibility that differences in the extraembryonic membranes might explain the interpopulation variation in embryonic acid tolerance found in this and earlier studies. Embryos were raised from fertilization to hatching at three different pH levels (pH 4.0, 4.25, and 7.5) in the laboratory, and acid stress tolerance was measured in terms of embryonic survival, growth and development (i.e., size and age at hatching). The results show that the higher acid tolerance of acid population embryos (in terms of survival) was maternally determined, indicating adaptive maternal effects. The jelly removal experiment revealed that adaptation to acidity in embryonic survival may arise through variation related to structure/composition of the egg capsules. There was no evidence for a genetic basis in acid tolerance in sublethal effects, but additive and nonadditive genetic effects were found in embryonic growth and development, independently of treatment. The results indicate a role for maternal effects in local adaptation to acidity in amphibians, and genetically based differences in early life-histories among the populations.     

Geographic variation in acid stress tolerance of the moor frog,Rana arvalis. I. Local adaptation

Abstract Spatially varying directional selection together with restricted gene flow among populations is expected to lead to local adaptation. One environmental factor that potentially causes strong directional selection, but is little explored in evolutionary terms, is naturally and anthropogenically induced acidity. We studied local adaptation to acidity in four Swedish populations (two originating from areas that have suffered from severe anthropogenic acidification during the 1900s and two from areas which have remained neutral due to higher buffering capacity) of the moor frog Rana arvalis in a laboratory experiment by investigating whether differences in acid tolerance correspond to population origin. Embryos were raised from fertilization to hatching at three different pH levels (pH 4.0, 4.25 and 7.5), corresponding to levels experienced by these populations in nature, and acid stress tolerance was measured in terms of embryonic survival, hatchling size, and age. Evidence for local adaptation in all of these traits was found, the acid origin embryos having higher survival and less impaired growth performance under acid conditions than the neutral origin embryos. Our estimated rates of divergence (0.007–0.102 haldanes) suggest a rapid adaptation process in response to anthropogenic environmental change, and that the different traits have evolved at relatively similar rates.     

Molecular phenotyping of maternally mediated parallel adaptive divergence within Rana arvalis and Rana temporaria

When similar selection acts on the same traits in multiple species or populations, parallel evolution can result in similar phenotypic changes, yet the underlying molecular architecture of parallel phenotypic divergence can be variable. Maternal effects can influence evolution at ecological timescales and facilitate local adaptation, but their contribution to parallel adaptive divergence is unclear. In this study, we (i) tested for variation in embryonic acid tolerance in a common garden experiment and (ii) used molecular phenotyping of egg coats to investigate the molecular basis of maternally mediated parallel adaptive divergence in two amphibian species (Rana arvalis and Rana temporaria). Our results on three R. arvalis and two R. temporaria populations show that adaptive divergence in embryonic acid tolerance is mediated via maternally derived egg coats in both species. We find extensive polymorphism in egg jelly coat glycoproteins within both species and that acid‐tolerant clutches have more negatively charged egg jelly – indicating that the glycosylation status of the jelly coat proteins is under divergent selection in acidified environments, likely due to its impact on jelly water balance. Overall, these data provide evidence for parallel mechanisms of adaptive divergence in two species. Our study highlights the importance of studying intraspecific molecular variation in egg coats and, specifically, their glycoproteins, to increase understanding of underlying forces maintaining variation in jelly coats.     

Adaptive divergence in moor frog (Rana arvalis) populations along an acidification gradient: inferences from Q(st) -F(st) correlations

Microevolutionary responses to spatial variation in the environment seem ubiquitous, but the relative role of selection and neutral processes in driving phenotypic diversification remain often unknown. The moor frog (Rana arvalis) shows strong phenotypic divergence along an acidification gradient in Sweden. We here used correlations among population pairwise estimates of quantitative trait (P(ST) or Q(ST) from common garden estimates of embryonic acid tolerance and larval life-history traits) and neutral genetic divergence (F(ST) from neutral microsatellite markers), as well as environmental differences (pond pH, predator density, and latitude), to test whether this phenotypic divergence is more likely due to divergent selection or neutral processes. We found that trait divergence was more strongly correlated with environmental differences than the neutral marker divergence, suggesting that divergent natural selection has driven phenotypic divergence along the acidification gradient. Moreover, pairwise P(ST) s of embryonic acid tolerance and Q(ST) s of metamorphic size were strongly correlated with breeding pond pH, whereas pairwise Q(ST) s of larval period and growth rate were more strongly correlated with geographic distance/latitude and predator density, respectively. We suggest that incorporating measurements of environmental variation into Q(ST) -F(ST) studies can improve our inferential power about the agents of natural selection in natural populations.     

Effects of habitat fragmentation and road density on the distribution pattern of the moor frog Rana arvalis

1. The effects of habitat fragmentation on the distribution pattern of the moor frog Rana arvalis were investigated. Also, the possible isolation effects of the road network were taken into account.
2. Indications were found that habitat fragmentation partly explains the distribution pattern of the moor frog. The statistical models showed a positive effect of pond size (or marsh area) and a negative effect of road density on the probability of occupation of a moorland pond.
3. Because of the strong correlation between habitat quality variables and isolation variables, no unambiguous effects of isolation, described as the amount of suitable terrestrial habitat (moorland) in the surroundings of a moorland pond in a radius of 100–2000 m, could be demonstrated.
4. Spatial differences in road density can play a role in the selection of optimal locations for nature protection areas. The regression model used in this study predicts a reduced occupation probability in 55% of the study area. In the part of the study area adjacent to a motorway, occupation probability is lowered to less than 30%.
5. European studies of habitat fragmentation on amphibian species revealed a mean distance between occupied ponds of <1 km in all studies. This could be a general rule of thumb for persistent amphibian populations. Effects of pond size on the probability of occupation were more variable.
6. When discussing the effects of habitat fragmentation on amphibians and other ground dwelling species, the negative effects of roads are often underestimated.     

Microsatellite variation and population structure of the moor frog (Rana arvalis) in Scandinavia

Several recent studies have found amphibian populations to be genetically highly structured over rather short geographical distances, and that the rate of genetically effective dispersal may differ between the sexes. However, apart from the common frog ( Rana temporaria ) little is known about the genetic structuring and sex-biased dispersal in northern European amphibians. We investigated the patterns of genetic diversity and differentiation within and among Scandinavian populations of the moor frog ( Rana arvalis ) using microsatellite markers. The genetic diversity within local R. arvalis populations was not a simple linear negative function of latitude but a convex one: genetic diversity peaked in mid-latitude populations, and declined thereafter dramatically towards the north. The average degree of genetic differentiation among populations ( F _ST = 0.14) was lower than that observed for the common frog ( F _ST = 0.21), though the pattern of isolation by distance was similar for both species. Contrary to common frogs, no evidence for female-biased dispersal was found. The results reinforce the view that amphibian populations are—in general—highly structured over relatively small geographical distances, even in comparatively recently colonized areas.     

Morphometric differentiation of the moor frog (Rana arvalis Nilss.) in Central Europe

Morphometric variation of 11 characters was studied in 14 samples of Rana arvalis from allopatric population groups in Central Europe, representing two subspecies formerly recognised: R. a. arvalis and R. a. wolterstorffi. All samples from Poland (nine) were collected from the area, which is believed to be populated by the nominal form. In addition, the isolated population of R. arvalis from the Eastern Carpathians of Romania was classified to that form. All the Hungarian samples fall into the range of R. a. wolterstorffi. No significant differences in body size between sexes and among the populations from the three geographic regions (Poland, Hungary and Romanian Eastern Carpathians) were found. Multivariate analysis of the differences in body shape among groups showed that the populations from Hungary differed from the nominal form mainly in the relative hindlimb length. However, substantial overlap in overall shape differences resulted in a relatively low percentage of correct classifications to the respective geographic groups in the Discriminant Analysis. Large intrapopulation variation in the values of the two ratios (SVL/TL and TL/IMT) formerly used for differentiate the two European subspecies of R. arvalis makes them unreliable characters for distinguishing the two forms. The pattern of morphometric variation does not match the overall genetic divergence of the R. arvalis populations, which suggests that the body shape differences in this species result from the phenotypic plasticity correlated with local climatic factors.     

The sensitivity of the moor frog Rana arvalis Nilss. tadpoles to natural L-amino acids and its alteration in ontogeny

The Rana arvalis tadpoles at 27-40 stages responded to solution of some L-amino acids in concentration 10(-2) mol/l with feeding reactions. The range of perceptible amino acids increased during development from 10 to 16. The best feeding stimuli were aspartic and glutamic acids; amino-acids (proline and oxyproline), methionine, leucine and isoleucine did not evoke the significant reactions. The sensitivity to some amino acids (alanine, glutamine, lysine, ornithine, proline and valine) was 10(-5)-10(-4) mol/l at earlier stages and increased to 10(-6)-10(-5) mol/l during larval ontogenesis.     

Latitudinal divergence of common frog (Rana temporaria) life history traits by natural selection: evidence from a comparison of molecular and quantitative genetic data

The relative roles of natural selection and direct environmental induction, as well as of natural selection and genetic drift, in creating clinal latitudinal variation in quantitative traits have seldom been assessed in vertebrates. To address these issues, we compared molecular and quantitative genetic differentiation between six common frog (Rana temporaria) populations along an approximately 1600 km long latitudinal gradient across Scandinavia. The degree of population differentiation (QST approximately 0.81) in three heritable quantitative traits (age and size at metamorphosis, growth rate) exceeded that in eight (neutral) microsatellite loci (FST = 0.24). Isolation by distance was clear for both neutral markers and quantitative traits, but considerably stronger for one of the three quantitative traits than for neutral markers. QST estimates obtained using animals subjected to different rearing conditions (temperature and food treatments) revealed some environmental dependency in patterns of population divergence in quantitative traits, but in general, these effects were weak in comparison to overall patterns. Pairwise comparisons of FST and QST estimates across populations and treatments revealed that the degree of quantitative trait differentiation was not generally predictable from knowledge of that in molecular markers. In fact, both positive and negative correlations were observed depending on conditions where the quantitative genetic variability had been measured. All in all, the results suggest a very high degree of genetic subdivision both in neutral marker genes and genes coding quantitative traits across a relatively recently (< 9000 years) colonized environmental gradient. In particular, they give evidence for natural selection being the primary agent behind the observed latitudinal differentiation in quantitative traits.     

A quantitative genetic test of adaptive decoupling across metamorphosis for locomotor and life-history traits in the pacific tree frog, Hyla regilla

Metamorphosis is assumed to be beneficial because it can break developmental links between traits in the different phases of a complex life-cycle and thereby allow larval and adult phases to adapt independently. I tested the prediction that correlations between the larval and adult phases are smaller than within stages. I estimated phenotypic and additive genetic variances and correlations for tadpole swimming speed, frog jump distance, body size, and larval period in a single population of the Pacific tree frog, Hyla regilla. These traits are known or reasonably assumed to be important for survival in this and other anuran species from temporary ponds. Only the three size variables were affected by sire identity. Heritabilities for locomotor performance, larval period, and size-independent performance were low (0.00-0.23) and not significant. Body size measurements showed somewhat higher and statistically significant heritabilities (0.24-0.34). Most traits were phenotypically correlated. On average, phenotypic correlations were larger between phases than within phases (0.41 vs. 0.28). Genetic correlations involving body-size traits were positive and large, and average within- and between-phase genetic correlation coefficients were identical (0.81). These results do not support the adaptive decoupling hypothesis, and they indicate that a paucity of additive genetic variation is a likely constraint on the evolution of traits measured for this population.     

The role of genetic structure in the adaptive divergence of populations experiencing saltwater intrusion due to relative sea‐level rise

Saltwater intrusion into estuaries creates stressful conditions for nektonic species. Previous studies have shown that Gambusia affinis populations with exposure to saline environments develop genetic adaptations for increased survival during salinity stress. Here, we evaluate the genetic structure of G. affinis populations, previously shown to have adaptations for increased salinity tolerance, and determine the impact of selection and gene flow on structure of these populations. We found that gene flow was higher between populations experiencing different salinity regimes within an estuary than between similar marsh types in different estuaries, suggesting the development of saline‐tolerant phenotypes due to local adaptation. There was limited evidence of genetic structure along a salinity gradient, and only some of the genetic variation among sites was correlated with salinity. Our results suggest limited structure, combined with selection to saltwater intrusion, results in phenotypic divergence in spite of a lack of physical barriers to gene flow.     

Matriline effects on metamorphic traits in a natural system in the European common frog (Rana temporaria)

Successful reproduction is an important determinant of the fitness of an individual and of the dynamics of populations. Offspring of the European common frog (Rana temporaria) exhibit a high degree of variability in metamorphic traits. However, environmental factors alone cannot explain this phenotypic variability, and the influence of genetic factors remains to be determined. Here, we tested whether the maternal genotype influences developmental time, body size, and body condition of offspring in a forest pond in Germany. We collected fertilized eggs from all 57 clutches deposited in the pond. We used multilocus genotypes based on seven microsatellite loci to assign metamorphosed offspring to mothers and to determine the number of fathers for a single matriline. We tested the influence of genetic effects in the same environment by comparing variability of metamorphic traits within and between full‐sib offspring grouped to matrilines and tested whether multiple paternity increases the variability of metamorphic traits in a single matriline. The variability in size and body condition was higher within matrilines than between them, which indicates that these traits are more strongly influenced by environmental effects, which are counteracting underlying genetic effects. The developmental time varied considerably between matrilines and variability increased with the effective number of fathers, suggesting an additive genetic effect of multiple paternity. Our results show that metamorphic traits are shaped by environmental as well as genetic effects.     

The genetic basis of quantitative variation in susceptibility of Arabidopsis thaliana to Pseudomonas syringae (Pst DC3000): evidence for a new genetic factor of large effect

* Pathogens represent an important threat to plant communities and agriculture, and can shape many aspects of plant evolution. Natural variation in plant disease susceptibility is typically quantitative, yet studies on the molecular basis of disease resistance have focused mainly on qualitative variation. * Here we investigated the genetic architecture of quantitative susceptibility to the bacterium Pseudomonas syringae by performing a quantitative trait locus (QTL) analysis on the F2 progeny of two natural accessions of Arabidopsis thaliana under two nutrient treatments. * We found that a single QTL explains most of the variation (77%) in susceptibility between accessions Columbia (Col-0) and San Feliu-2 (Sf-2), and its effect is independent of nutrients. The Sf-2 allele at this QTL is dominant and can reduce the bacterial population size by 31-fold, much like a classical resistance (R) gene. However, minor QTLs, whose effects are altered by nutrient treatment, were also detected. * Surprisingly, we found that none of the QTLs for susceptibility had any effect on fruit production, suggesting that the use of resistance genes for crop improvement and evolutionary analysis of plant-pathogen interactions requires caution.     

Local Adaptation and Genetics of Acid-Stress Tolerance in the Moor Frog, Rana arvalis

As potential to adapt to environmental stress can be essential for population persistence, knowledge on the genetic architecture of local adaptation is important for conservation genetics. We investigated the relative importance of additive genetic, dominance and maternal effects contributions to acid stress tolerance in two moor frog (Rana arvalis) populations originating from low and neutral pH habitats. Experiments with crosses obtained from artificial matings revealed that embryos from the acid origin population were more tolerant to low pH than embryos from the neutral origin population in embryonic survival rates, but not in terms of developmental stability, developmental and growth rates. Strong maternal effect and small additive genetic contributions to variation were detected in all traits in both populations. In general, dominance contributions to variance in different traits were of similar magnitude to the additive genetic effects, but dominance effects outweighed the additive genetic and maternal effects contributions to early growth in both populations. Furthermore, the expression of additive genetic variance was independent of pH treatment, suggesting little additive genetic variation in acid stress tolerance. The results suggest that although local genetic adaptation to acid stress has taken place, the current variation in acid stress tolerance in acidified populations may owe largely to non-genetic effects. However, low but significant heritabilities (h ² 0.07–0.22) in all traits – including viability itself – under a wide range of pH conditions suggests that environmental stress created by low pH is unlikely to lower moor frog populations' ability to respond to selection in the traits studied. Nevertheless, acid conditions could lower populations' ability to respond to selection in the long run through reduction in effective population size.     

Geographic variation as a result of evolution of the traits with broad and narrow norms of reaction in the moor frog (Rana arvalis)

Females reproductive, size, and age characteristics were studied in isolated local populations of Rana arvalis in the southern and northern parts of its range. The yearlings of the southern populations used to get larger by their first overwintering due to earlier beginning of the breeding season, as compared with the yearlings of the northern population. As a result, "southern" females become sexually mature at the age of two years while the "northern" ones become mature at the age of three years. This causes geographic differences in age composition among two populations, the "southern" reproductive females being younger on average than the "northern" ones. The earlier female maturation in the first case is not compensated by respective rise of the growth rate; to the contrary, the "southern" females grow more slowly during the first two years of their life and appear to be smaller than the "norhern" ones. These reproduction and growth patterns arise supposedly due to paedomorphosis, which causes specific reproductive characteristics, namely decrease in the egg size, increase in the reproductive effort and more strong correlation between female fertility and body size. Local and geographic differences are expressed not in the extent but in the structure of reproductive pattern, as no negative correlation was revealed between female reproductive age and body size in the southern populations. Southern habitats cannot be considered as "unfavourable with respect to body size", so the geographic differences under consideration cannot be explained by optimization of the reproductive strategies at population level. Paedomorphosis appears as a result of the female maturation rate possessing a wider norm of reaction than the growth rate. At the same time, fixation of the specific growth rate narrows norm of reaction of some other characters important for the phenotype reproductive fitness thus predetermining their subsequent evolution.     

Sex-biased genetic component distribution among populations: additive genetic and maternal contributions to phenotypic differences among populations of Chinook salmon

An approach frequently used to demonstrate a genetic basis for population-level phenotypic differences is to employ common garden rearing designs, where observed differences are assumed to be attributable to primarily additive genetic effects. Here, in two common garden experiments, we employed factorial breeding designs between wild and domestic, and among wild populations of Chinook salmon (Oncorhynchus tshawytscha). We measured the contribution of additive (V(A)) and maternal (V(M)) effects to the observed population differences for 17 life history and fitness-related traits. Our results show that, in general, maternal effects contribute more to phenotypic differences among populations than additive genetic effects. These results suggest that maternal effects are important in population phenotypic differentiation and also signify that the inclusion of the maternal source of variation is critical when employing models to test population differences in salmon, such as in local adaptation studies.     

Maxillary sinus variation in hybrid macaques: implications for the genetic basis of craniofacial pneumatization

There has been a long‐standing debate regarding the diversification of paranasal sinuses, namely pneumatized spaces in the face. Functional adaptation and structural constraints have generally been suggested to explain sinus diversification in vertebrates. Here we investigated variation in the maxillary sinus and the external facial cranium in hybrid Taiwanese–Japanese macaques to estimate the genetic basis of phenotypic differences. The Taiwanese macaques have a large sinus, whereas the Japanese macaques have a small sinus; they are also significantly different in their external craniofacial morphology. Variations in the hybrids' external craniofacial morphology can be mostly explained by a simple additive model. In contrast, their sinus morphology significantly deviates from the value expected under this additive model, wherein most hybrids have a large sinus, similar to that in Taiwanese macaques, regardless of the degree of hybridization. When the whole structure is considered, a novel phenotype can be seen in the hybrids. Our results suggest that the sinus and face are independent of each other, both genetically and developmentally, and that the small sinus is mainly caused by intrinsic genetic factors, rather than being structurally constrained by the craniofacial architecture. Such genetic factors may have contributed to the enigmatic diversity of craniofacial pneumatization. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 333–347.     

History vs. current demography: explaining the genetic population structure of the common frog (Rana temporaria)

The amount of genetic variability at neutral marker loci is expected to decrease, and the degree of genetic differentiation among populations to increase, as a negative function of effective population size. We assessed the patterns of genetic variability and differentiation at seven microsatellite loci in the common frog (Rana temporaria) in a hierarchical sampling scheme involving three regions (208-885 km apart), three subregions within regions and nine populations (5-20 km apart) within subregions, and related the variability and differentiation estimates to variation in local population size estimates. Genetic variability within local populations decreased significantly with increasing latitude, as well as with decreasing population size and regional site occupancy (proportion of censured localities occupied). The positive relationship between population size and genetic variability estimates was evident also when the effect of latitude (cf. colonization history) was accounted for. Significant genetic differentiation was found at all hierarchical levels, and the degree of population differentiation tended to increase with increasing latitude. Isolation by distance was evident especially at the regional sampling level, and its strength increased significantly towards the north in concordance with decreasing census and marker-based neighbourhood size estimates. These results are in line with the conjecture that the influence of current demographic factors can override the influence of historical factors on species population genetic structure. Further, the observed reductions in genetic variability and increased degree of population differentiation towards the north are in line with theoretical and empirical treatments suggesting that effective population sizes decline towards the periphery of a species' range.